Analysis of RNA by primer extension method
Analysis of RNA by primer extension method
Primer extension is primarily used for mRNA 5' end mapping. poly (A) + RNA is first hybridized to an excess of a 5' end-labeled single-stranded oligonucleotide primer that is complementary to the target RNA, and then the primer is extended with reverse transcriptase. The resulting cDNA is complementary to the RNA template and is equal in length to the distance between the 5' end of the primer and the 5' end of the RNA. This experiment is based on the "Guide to Molecular Cloning, Third Edition", translated by Huang Peitang et al.
Operation method
Analysis of RNA by primer extension method
Principle
Primer extension is primarily used for mRNA 5' end mapping. poly (A)+RNA is first hybridized to an excess of 5' end-labeled single-stranded oligonucleotide primer that is complementary to the target RNA, and then the primer is extended with reverse transcriptase. The resulting cDNA is complementary to the RNA template and is equal in length to the distance between the 5' end of the primer and the 5' end of the RNA.
Materials and Instruments
Polynucleotide Kinase RNAase Protein Inhibitor Reverse Transcriptase Oligonucleotide Primer ATP Move I. Materials For more product details, please visit Aladdin Scientific website.
Ammonium acetate Chloroform DTT Ethanol Formamide spiking buffer KCl Phenol Primer extension reaction mixture Sodium acetate TE Trichloroacetic acid
Denaturing polyacrylamide gel Water bath Whatman 3 MM filter paper
1. Buffers and solutions
Ammonium acetate (10 mol/L)
Chloroform
DTT ( 1 mol/L)
Ethanol
Formamide spiking buffer (80% deionized formamide, 10 mmol/L EDTA (pH 8.0), 1 mg/ml xylene cyanide blue, 1 mg/ml bromophenol blue)
KCl ( 1.25 mol/L )
Phenol
Primer extension reaction mixture (20 mmol/L Tris-Cl (pH 8.4) (room temperature), 10 mmol/L MgCl2, 1.6 mmol/L dNTP solution, 50 μg/ml actinomycin D)
Sodium acetate (3 mol/L, pH 5.2)
TE ( pH 7.6)
Trichloroacetic acid (1% and 10% TCA )
2. Enzyme and buffer
Polynucleotide kinase
RNAase protein inhibitors
Reverse transcriptase
3. gels
Denaturing polyacrylamide gel with 8 mol/L urea
4. nucleic acids and oligonucleotides
Vector RNA (yeast tRNA)
Radiolabeled DNA relative molecular mass reference for gel electrophoresis
RNA to be analyzed
Oligonucleotide primers
5. Radioactive compounds
[ γ-32P ] ATP (10 mCi/ml, 7000 Ci/mmol)
6. Special equipment
42~95°C ( including appropriate recharacterization temperature) water bath
Whatman 3 MM filter paper (or equivalent)
II. Methods
Preparation of Oligonucleotide Probes
1. Phosphorylate the oligonucleotide in the following system.
Oligonucleotide primer (5-7 pmol or 60 ng) 1 μl
Deionized water 6.5 μl
10X kinase buffer 1.5 μl
Polynucleotide kinase (~10 units) 1 μl
[ λ-32P ] ATP (7000 Ci/mmol) 2 μl
Incubate at 37℃ for 60 min.
2. Add 500 ml TE (pH 7.6) to terminate the reaction. Add 25 μg of carrier RNA.
3. Add 400 μl of equilibrated phenol (pH 8.0) and 400 μl of chloroform (or 800 μl of a 1:1 mixture of phenol and chloroform) and shake vigorously for 20 s. Centrifuge for 2 min to separate the aqueous and organic phases. Shake vigorously for 20 s. Centrifuge for 2 min to separate the aqueous and organic phases.
4. Transfer the aqueous layer to a new sterile centrifuge tube, extract with 800 μl of chloroform and shake vigorously for 20 s. Centrifuge for 2 min to separate the aqueous and organic phases. Transfer the aqueous layer to a new sterile centrifuge tube.
5. Repeat step 4.
6. Add 55 μl of sterile 3 mol/L sodium acetate (pH 5.2) and 1 ml of ethanol to the aqueous layer from step 5. Mix well and leave at -70°C for at least 1 h. Repeat step 4.
7. Centrifuge at maximum speed for 15 min to collect the oligonucleotide primer precipitate and discard the radioactive supernatant. Wash again with 70% ethanol, discard the supernatant and air dry the precipitate. Dissolve the precipitate in 500 μl TE (pH 7.6).
8. Count 2 μl of radiolabeled oligonucleotide primers in 10 ml of flashing solution in a liquid flash counter. Assume 80% recovery to calculate primer-specific radioactivity. The primer-specific activity should be 2X106 cpm/pmol.
Hybridization and Extension of Oligonucleotide Primers
9. Mix 104~105 cpm (20~40 fmol) of DNA primer and 0.5~150 μg of RNA to be analyzed. Add 0.1x volume of 3 mol/L sodium acetate (pH 5.2) and 2.5x volume of ethanol. Place at -70°C for 60 min and centrifuge at 4°C for 10 min at maximum speed to recover the RNA. Wash the precipitate with 70% ethanol and centrifuge again. Carefully remove the ethanol and leave at room temperature until the last trace of ethanol has evaporated.
10. Resuspend the precipitate with 8 μl of TE (pH 7.6) per tube and aspirate several times to dissolve it.
11. Add 2.2 μl of 1.25 moI/L KCl. mix gently and centrifuge for 2 s to settle the liquid to the bottom of the tube.
12. Place the oligonucleotide/RNA mixture in a water bath at the appropriate recovery temperature. Incubate for 15 min at the optimal temperature as determined by the pre-test.
13. While the oligonucleotides and RNA are being replicated, add DTT and reverse transcriptase to the primer extension mixture as follows: Dissolve 300 μl of the primer extension mixture on ice, then add 3 μl of 1 mol/L DTT and reverse transcriptase at a final concentration of 1 to 2 units/μl. Add 0.1 unit/μl of protein inhibitor of RNAase, invert several times and mix gently, keep on ice.
14. Remove the tube containing the oligonucleotide primer and RNA from the water bath and centrifuge for 2 s to settle the liquid to the bottom of the tube.
15. Add 24 μl of primer extension mixture to each tube. Mix gently and centrifuge to bring the liquid to the bottom of the tube.
16. Incubate at 42°C for 1 h to allow the primer extension reaction to proceed.
17. Add 200 μl TE (pH 7.6), 100 μl equilibrium phenol (pH 8.0) and 100 μl chloroform to terminate the reaction. Shake for 20 s and centrifuge for 4 min at room temperature to separate the aqueous and organic phases.
18. Add 50 μl of 10 mol/L ammonium acetate and 700 μl ethanol to precipitate nucleic acid. Mix well with shaking and place at -70°C for at least 1 h. Purification and analysis of the primer extension products.
19. Centrifuge at 4°C for 10 min to recover the precipitated nucleic acid and carefully wash with 400 μl of 70% ethanol. centrifuge at 4°C for 5 min and remove the 70% ethanol wash solution with a pipette tip. Leave at room temperature until the last visible ethanol has evaporated.
20. Dissolve the nucleic acid precipitate with 10 μl of amyl formate sampling buffer, repeatedly aspirating to aid redissolution.
21. Heat the sample at 95°C for 8 min and place on an ice bath. Immediately analyze the primer extension products by denaturing polyacrylamide gel electrophoresis.
22. When the tracer dye has migrated the appropriate distance in the gel, turn off the power and open the lid of the electrophoresis unit. Gently pry off one side of the larger glass plate and slowly remove the plate from the gel. Cut off a corner of the gel to orient it.
23. If the polyacrylamide gel used is 1 mm thick, fix it in the TCA. Transfer the glass plate containing the gel to a tray containing an excess of 10% TCA and gently shake or rotate the tray for 10 min at room temperature.
24. Pour off the 10% TCA and refill with an excess of 1% TCA and gently shake or rotate the tray for 5 min at room temperature.
25. Pour off 1% TCA and rinse the fixed gel with deionized water. Remove the glass plate and gel together from the tray and place on top of the platform with a paper towel to remove excess water from the edges.
26. Cut a piece of Whatman 3 MM filter paper (or equivalent) 1 cm larger than the gel on all sides. Lay the filter paper on top of the gel and invert the glass plate to transfer the gel onto the filter paper.
27. Remove the glass plate and dry the gel in a thermal vacuum gel dryer at 60°C for 1.0 to 1.5 h. The gel should be dried in a vacuum gel dryer.
28. The image of the gel was obtained by radiographic autoradiography or phosphorimaging.